Endophytic Fungi: An Effective Alternative Source of Plant-Derived Bioactive Compounds for Pharmacological Studies

Plant-associated fungi (endophytic fungi) are a biodiversity-rich group of microorganisms that are normally found asymptomatically within plant tissues or in the intercellular spaces. Endophytic fungi promote the growth of host plants by directly producing secondary metabolites, which enhances the plant’s resistance to biotic and abiotic stresses. Additionally, they are capable of biosynthesizing medically important “phytochemicals” that were initially thought to be produced only by the host plant. In this review, we summarized some compounds from endophyte fungi with novel structures and diverse biological activities published between 2011 and 2021, with a focus on the origin of endophytic fungi, the structural and biological activity of the compounds they produce, and special attention paid to the exploration of pharmacological activities and mechanisms of action of certain compounds. This review revealed that endophytic fungi had high potential to be harnessed as an alternative source of secondary metabolites for pharmacological studies.


Introduction
The term "endophytic fungi" refers to fungi that live in plant tissues throughout the entire or partial life cycle by establishing a mutually beneficial symbiotic relationship with its host plant without causing any adverse effect or disease [1,2]. They are natural components of the plant micro-ecosystem that positively affect the physiological activities of the host plant in several ways, including producing hormones such as indoleacetic acid, biosynthesizing and acquiring nutrients for plant growth and development, secreting stress-adaptor metabolites to protect the host plant from the invasion of herbivores, pathogens, and improving the host's adaptability to abiotic stressors. In return, plants provide habitats and nutrients for endophytic fungi [3,4]. Endophytic fungi are capable of producing a rich variety of bioactive substances and can produce compounds that are identical or similar to pharmacological activities identified from plants [5]. They produce a range of metabolites of different chemical classes, including alkaloids, flavonoids, steroids, terpenoids, and phenolic compounds. Some compounds show pleiotropic and interesting pharmacological activities, including antimicrobial, antioxidant, anti-diabetic, anti-malarial, and antitumor properties. The discovery of these structurally novel and diverse active compounds provides a valuable resource for studying natural medical products from the microbiome [6][7][8]. In the search for bioactive molecules as pro-drug compounds or in 1 Lophiostoma sp. Eucalyptus exserta Guangzhou, China. Scorpinone Antibacterial [22] 2 Mycosphaerella sp.

α-Pyrones
Two tetrasubstituted α-pyrone derivatives-Neurospora udagawae udagawanones A-B 11-12 ( Figure 3)-were isolated from oak endophytic fungi, with both containing unique oxidation functional groups at the C-2 position. Compound 11 exhibited potent antifungal activity against Rhodoturula glutinis with MIC = 66 µg/mL). Additionally, compounds 11 and 13 showed moderate cytotoxic activity against KB3.1 cells with IC 50 = 27 µg/mL [40]. The study revealed moderate activity of compounds 11 and 12 against fungi and mammalian cells, and this may be as a result of the method (serial dilution antimicrobial assay) used; therefore, it is suggested that other biological tests be employed to verify these findings. The nigerapyrones A-B 13-14 ( Figure 3) were obtained from Aspergillus niger MA-132, which was isolated from the mangrove plant Avicennia marina. Compounds 13-14 both showed potent antifungal activities against two tumor cell lines (HL60 and A549), with IC 50 values ranging from 0.3 to 5.41 µM [41]. The ficipyrones A-B 15-16 ( Figure 3) were isolated from solid cultures of Pestalotiopsis fici. Compound 15 showed significant antifungal activity against Gibberella zeae CGMCC 3.2873, with an IC 50 value of 15.9 µM, but had no activity against Fusarium culmorum CGMCC 3.4595 and Verticillium aiboatrum CGMCC 3.4306 [42].

Other Polyketides
The phomaketides A-E 22-26 ( Figure 4), pseurotins A3 27 (Figure 4), and pseurotins G 28 ( Figure 4) were isolated from fermentation broth and mycelial extracts of the marine red algae endophytic fungus Phoma sp. NTOU4195. The mouse macrophages RAW264 were induced using the endothelial progenitor cells of human umbilical cord blood, lipopolysaccharide (LPS), to assess the anti-angiogenic and anti-inflammatory activities of all compounds. Compound 22 showed potent anti-angiogenic activity by inhibiting endothelial cell proliferation, with an IC50 value of 8.1 μM. Compound 24 at the concentration of 20 μM induced effective nitric oxide (NO) inhibition activity against LPS-induced RAW264.7 cells, with an IC50 value of 8.8 μM [45]. There were two tetracyclic polyketide compounds, simplicilone A-B 29-30 (Figure 4), containing helical centers obtained from the broth culture of the endophytic fungus Simplicillium sp., which was isolated from the bark of the medicinal plant Duguetia staudtii (Engl. and Diels) Chatrou in the Cameroon region. Compounds 29-30 showed weak cytotoxic activities against the KB3.1 cell line, with IC50 values of 1.25 μg/mL and 2.29 μg/mL, respectively, but had no antimicrobial activity against the tested bacteria (Staphylococcus aureus DSM 346 and Bacillus subtilis

Other Polyketides
The phomaketides A-E 22-26 ( Figure 4), pseurotins A 3 27 (Figure 4), and pseurotins G 28 ( Figure 4) were isolated from fermentation broth and mycelial extracts of the marine red algae endophytic fungus Phoma sp. NTOU4195. The mouse macrophages RAW264 were induced using the endothelial progenitor cells of human umbilical cord blood, lipopolysaccharide (LPS), to assess the anti-angiogenic and anti-inflammatory activities of all compounds. Compound 22 showed potent anti-angiogenic activity by inhibiting endothelial cell proliferation, with an IC 50 value of 8.1 µM. Compound 24 at the concentration of 20 µM induced effective nitric oxide (NO) inhibition activity against LPS-induced RAW264.7 cells, with an IC 50 value of 8.8 µM [45]. There were two tetracyclic polyketide compounds, simplicilone A-B 29-30 (Figure 4), containing helical centers obtained from the broth culture of the endophytic fungus Simplicillium sp., which was isolated from the bark of the medicinal plant Duguetia staudtii (Engl. and Diels) Chatrou in the Cameroon region. Compounds 29-30 showed weak cytotoxic activities against the KB3.1 cell line, with IC 50 values of 1.25 µg/mL and 2.29 µg/mL, respectively, but had no antimicrobial activity against the tested bacteria (Staphylococcus aureus DSM 346 and Bacillus subtilis DSM 10) [46]. 5R-hydroxyrecifeiolide 31 (Figure 4), 5S-hydroxyrecifeiolide 32 (Figure 4), and ent-cladospolide F-H 33-35 ( Figure 4) were also isolated from the endophytic fungal strain Cladosporium cladosporioides MA-299, which was obtained from the leaves of the mangrove plant Bruguiera gymnorrhiza from Hainan Island, China. Compounds 31-35 showed potent antimicrobial activities against Escherichia coli and Staphylococcus aureus, with MIC values ranging from 1.0 to 64 µg/mL. Compound 33 showed moderate inhibition activity against acetylcholinesterase, with an IC 50 value of 40.26 µM [47]. The antimicrobial polyketide compound, palitantin 36 ( Figure 4), was obtained from Aspergillus fumigatiaffnis and isolated from healthy leaves of Tribulus terrestris L. In addition, compound 36 showed effective antimicrobial activity against the multi-drug-resistant pathogens Enterococcus faecalis UW 2689 and Streptococcus pneumoniae 25697, both with an MIC value of 64 µg/mL [48]. The four polyketide derivatives-isotalaroflavone 37 ( Figure 4), (+/−)-50dehydroxytalaroflavone 38-39 ( Figure 4), and bialternacin G 40 ( Figure 4)-were obtained from the endophytic fungus Alternaria alternata ZHJG5 isolated from the leaves of Cercis chinensis, which was collected from the Nanjing Botanical Garden, Nanjing, China. They exhibited potent antimicrobial activity against Xanthomonas oryzae pv. oryzicola (Xoc) and Ralstonia solanacearum, with MIC values ranging from 0.5 to 64 µg/mL. Compound 37 at the concentration of 200 µg/mL showed a significant protective effect against the bacterial blight of rice caused by Xanthomonas oryzae pv. oryza, with a protection rate of 75.1% [49]. Four polyketide derivatives containing the benzoisoquinoline-9-one moiety structure peyronetides A-D 41-44 ( Figure 4) were isolated from the mycelial crude acetone extract of Peyronellaea sp. FT431. Compounds 41-42 showed moderate to weak cytotoxic activity against human kidney cancer cell line TK10 and human ovarian cancer cell line A2780cisR, with IC 50 values ranging from 6.7 to 29.2 µM [50]. The aromatic polyketide compound, (−)alternamgin 45 (Figure 4), was obtained from potato dextrose broth cultures of the endophytic fungus Alternaria sp. MG1 isolated from Vitis quinquangularis. This compound was of particular interest because it had the rare dibenzopyrone functionality of 6/6/6/6/5/6/6/6 heptacyclic backbone. Compound 45 displayed a weak cytotoxic activity against cells from two tested cell lines (Hela and HepG2), both with IC 50 values exceeding 20 µM [51].
In summary, Polyketides, such as chromones and α-pyrone, and their derivatives identified from plant sources have also been found in endophytic fungi in recent studies. Chromones and their derivatives isolated from both plant and endophytic fungi sources all showed antimicrobial properties against specific pathogens; therefore, chromones from endophytic fungus can be used in the development of antimicrobials in the place of plant chromones to reduce the depletion of plants' resources in the ecosystem.
The in vitro cytotoxicity of compound 94 was evaluated against three human tumor cell lines (A549, LOVO, and MCF-7), to which compound 94 showed weak cytotoxic activities against all human tumor cell lines, with IC 50 values of 76.83, 68.08, and 40.55 µg/mL, respectively [65]. The enantiomeric bromotyrosine alkaloids S-Acanthodendrilline 95 ( Figure 8) and R-Acanthodendrilline 96 ( Figure 8) were isolated from the ethyl acetate extract of the sponge endophytic fungus Acanthodendrilla sp. The cytotoxic activities of compounds 95-96 against human non-small cell lung cancer H292 and normal human immortalized fibroblast HaCaT cell lines were evaluated using the MTT method. Compound 95 (IC 50 value of 58.5 µM) was approximately three times more potent than compound 96 (IC 50 value of 173.5 µM) against the H292 cell line. Compounds 95-96 exhibited efficient and selective cytotoxic activities against H292 and HaCaT cell lines, with IC 50 values ranging from 58.5 to 173.5 µM and >400 µM, respectively [66]. Three phenylpyridone derivatives, citridones E-G 97-99 ( Figure 8), were obtained from the endophytic fungal strain Penicillium sumatrense GZWMJZ-313 9, which was isolated from the leaves of Garcinia multiflora. These compounds showed moderate to weak antimicrobial activities against Staphylococcus aureus ATCC6538, Pseudomonas aeruginosa ATCC10145, and Escherichia coli ATCC11775, with MIC values ranging from 32 to 128 µg/mL [67]. Two isoprenylisoindole alkaloids, diaporisoin-

Diketopiperazine Derivatives
The thiodiketopiperazine alkaloid, phaeosphaones D 77 (Figure 7), featuring an unusual β-(oxy) thiotryptophan motif, was obtained from endophytic fungus Phaeosphaeria fuckelii isolated from the medicinal plant Phlomis umbrosa. Compound 77 showed stronger philia, Escherichia coli, Staphylococcus aureus, Vibrio arveyi, and V. parahaemolyticus). Compounds 86-90 showed potent antimicrobial activities against S. aureus, with MIC values ranging from 0.25 to 32 μg/mL [63]. Spirobrocazines A-C 91-93 ( Figure 7) were isolated from the mangrove-derived Penicillium brocae MA-231. Compounds 91-93 contained a 6/5/6/5/6 cyclic system with a rare spirocyclic center at C-2. All compounds showed moderate antimicrobial activities against S. aureus, Aeromonas hydrophilia, and Vibrio harveyi, with MIC values ranging from 16 to 64 μg/mL [64]. In a nutshell, anti-angiogenic and anti-inflammatory activities were the main activities of alkaloids in both plants and endophytic fungi. In addition, phomaketides and their derivatives that were isolated from fungal endophytes possess antimicrobial activity just as those isolated in plants; therefore, alkaloids producing endophytic fungi can be used in the development of anti-angiogenic, anti-inflammatory, and antimicrobial drugs for both human and animal use.

Sesquiterpenoids and Their Derivatives
The 1-methoxypestabacillin B 107 ( Figure 9) was obtained from brown rice cultures of endophytic fungus Diaporthe sp. SCSIO 41011 isolated from the stem of the mangrove plant Rhizophora stylosa. Compound 107 was evaluated for the reversal of HIV incubation period and anti-influenza A virus activities, to which compound 107 did not show antiviral activity. However, its structure could serve as the backbone for the synthesis of more potent antiviral compounds [69]. The eremophilane-type sesquiterpenoids rhizoperemophilanes A-N 102-115 ( Figure 9) were isolated from the ethyl acetate extract of Rhizopycnis vagum Nitaf22. Compound 111 contained a C-4/C-11 epoxide, and compound 115 had a 3-nor-eremophilane lactone-lactam skeleton. All compounds were evaluated for their cytotoxic activities against five tested human cancer cells (BGC823, Daoy, HCT116, HepG2, and NCI-H1650) and inhibition activities against radicle growth in rice seedlings. Compound 115 showed high selective cytotoxicity against NCI-H1650 and BGC823 cell lines, with IC50 values of 15.8 μM and 48.2 μM, respectively, while no significant cytotoxic activity was observed for other compounds at IC50 > 50 μm. Compounds 106-107 and 113-114 showed strong phytotoxic activities against radicle growth in rice seedlings at a concentration of 200 μg/mL, where the inhibition exceeded 50% [70]. The bisabolane-type sesquiterpene, trichoderic acid 116, (Figure 9) and acorane-type sesquiterpene, 2β-hydroxytrichoacorenol 117 (Figure 9), were obtained from Trichoderma sp. PR-35 culture, an endophytic fungus isolated from stems of Paeonia delavayi. Compounds 116-117 were tested for antimicrobial activity against two pathogens (Escherichia coli, and Shigella sonnei) using an agar diffusion method. Compounds 116-117 showed moderate to weak antimicrobial ac-

Diterpenoids
The ring diterpene diaporpenoid A 131 (Figure 10), containing a 5/10/5-fused tricyclic ring system, was isolated from the MeOH extract obtained from cultures of the mangrove endophytic fungus Diaporthe sp. QYM12. Compound 131 showed significant anti-inflammatory activity by inhibiting LPS-induced NO production in a mouse macrophage cell line RAW264.7, with an IC 50 value of 21.5 µM [75]. The pimarane-type diterpene Libertellenone M 132 ( Figure 10) was isolated from the marine source endophytic fungus Phomopsis sp. S12. Compound 132 inhibited pro-inflammatory cytokines IL1β and IL-18 mRNA expression in colon tissue, significantly reduced the cleavage of pro-caspase1, and dose-dependently inhibited the NF-κB nuclear translocation in macrophages. Clinical indications of acute colitis induced by 3% dextran sulphate sodium in mice were attenuated by intravenous administration of different doses of compound 132 (10 or 20 mg/kg), which is a potent inhibitor of NLRP3 inflammatory vesicles and may be a new medicine for treating acute colitis [76]. Three pimarane-type diterpenoids-pedinophyllol K 133 (Figure 10), pedinophyllol L 134 (Figure 10), and libertellenone T 135 ( Figure 10)-were isolated from the endophytic fungal Phomopsis sp. S12 culture using the OSMAC strategy. The antiinflammatory activities of all compounds were assessed using an LPS-induced inflammation model of mouse macrophage RAW264.7. Compound 135 dose-dependently inhibited the expression of inflammatory factors IL-1β and IL-6 at the mRNA level. Additionally, the anti-inflammatory activity of compounds 133-134 was similar to that of compound 135 in terms 0f IL-6 inhibition [77]. Two tetranorlabdane diterpenoids botryosphaerins G-H 136-137 ( Figure 10) were obtained from the ethyl acetate extract of Botryosphaeria sp. P483 isolated from the branches of the herb Huperzia serrata (Thunb.) Trev. and tested for their antifungal activities against Gaeumannomyces graminis, Fusarium solani, and Pyricularia oryzae by the disk diffusion method. Compound 137 showed effective antifungal activity at a concentration of 100 µg/disk with an inhibitory zone diameter of 9 mm. (The inhibitory zone diameter of positive control carbendazim was 15-18 mm.) Compounds 136-137 were evaluated for their nematicidal activities against Panagrellus redivivus and Caenorhabditis elegans and showed weak nematicidal activities, with 30% and 28% fatality rates at a 24h action concentration of 400 mg/L, respectively [78]. The isopimarane diterpene sphaeropsidin A 138 ( Figure 10 Figure 10), a diterpene containing a 6-5-6-6 ring system, was obtained from the endophytic fungus Trichoderma atroviride S361 of Cephalotaxus fortunei and was not tested for any biological activities [81]. Therefore, further studies are needed to identify the potential biological activity of this compound in the future. The new tetranorlabdane diterpenoids, asperolides A-

Triterpenoids
The 24-homo-30-nor-cycloartane triterpenoid 154 ( Figure 11) was isolated from the endophytic fungus Mycoleptodiscus indicus FT1137. Compound 154 showed no activity against the human ovarian cancer cell line A2780 at a concentration of 20 µg/mL [83]. Three Lanostane-type triterpenes-sclerodols A-B 144-145 ( Figure 11) and lanosta-8,23dien-3β,25-diol 146 ( Figure 11)-were obtained from Eucalyptus grandis cultures derived from the endophytic fungus Scleroderma UFSMSc1, and the antifungal activities of compounds 144-146 against Candida albicans and Candida parapsolosis were evaluated by the agar diffusion method. Compounds 144-146 showed moderate to weak antifungal activities, with MIC values ranging from 12.5 to 50 µg/mL. The antifungal effects of these compounds against C. albicans were associated with the inhibition of the selenocysteine methyltransferase (SMT) activity [84]. Fusidic acid 147 ( Figure 11) was obtained from the cultures of the endophytic fungus Acremonium pilosum F47, isolated from the stem of Mahonia fortunei using the bioactivity-guided assay, and the antimicrobial activities of compound 147 against four human pathogens were tested (S. aureus ATCC 6538, B. subtilis ATCC 9372, P. aeruginosa ATCC 27853, and E. coli ATCC 25922) and evaluated. Compound 147 showed effective antimicrobial activities against S. aureus ATCC 6538 and B. subtilis ATCC 9372. The acetylation of the C-16 hydroxyl group of compound 147 was essential for antimicrobial action [85]. Two new ring A-cleaved lanostane-type triterpenoids, glometenoid A-B 148-149 (Figure 11), were obtained from the ethyl acetate extract of the mason pine endophytic fungus Glomerella sp. F00244. The cytotoxic activity of compounds 148-149 against the human ovarian cancer cell line HeLa was tested using the MTT assay. Compound 148 showed weak cytotoxic activity at a concentration of 10 µM with 21% inhibition [83]. Nine highly oxygenated schitriterpenoids-kadhenrischinins A-H 150-157 ( Figure 11) and 7β-schinalactone C 158 ( Figure 11)-were isolated from Penicillium sp. SWUKD4.1850, and compounds 154-157 contained a unique 3-one-2-oxabicyclo [1-3]-octane motif. All compounds were tested for their cytotoxic activities against the HepG2 tumor cell lines using the MTT assay, and these compounds showed weak cytotoxic activities, with IC 50 values ranging from 14.3 to 40 µM [86]. Two tetracyclic triterpenoids-integracide E 159 ( Figure 11) and isointegracide E 160 ( Figure 11)-were isolated from the mycelia of Hypoxylon sp. 6269. Compound 159 showed weak inhibition activity against the HIV-1 integrase, with an IC 50 value of 31.63 µM [87]. The tetracyclic triterpenoids, integracides H-J 161-163 (Figure 11), were obtained from the endophytic fungus Fusarium sp., which was isolated from the roots of Mentha longifolia L. (Labiatae) and were evaluated for antileishmanial activity against L. donovani promastigotes. Compound 161 showed significant antileishmanial activity, with an IC 50

Diterpenoids
The ring diterpene diaporpenoid A 131 (Figure 10), containing a 5/10/5-fused tricyclic ring system, was isolated from the MeOH extract obtained from cultures of the mangrove endophytic fungus Diaporthe sp. QYM12. Compound 131 showed significant anti-inflammatory activity by inhibiting LPS-induced NO production in a mouse macrophage cell line RAW264.7, with an IC50 value of 21.5 μM [75]. The pimarane-type diterpene Libertellenone M 132 ( Figure 10) was isolated from the marine source endophytic fungus Phomopsis sp. S12. Compound 132 inhibited pro-inflammatory cytokines IL1β and IL-18 mRNA expression in colon tissue, significantly reduced the cleavage of pro-caspase1, and dose-dependently inhibited the NF-κB nuclear translocation in macrophages. Clinical indications of acute colitis induced by 3% dextran sulphate sodium in mice were attenuated by intravenous administration of different doses of compound 132 (10 or 20 mg/kg), which is a potent inhibitor of NLRP3 inflammatory vesicles and may be a new medicine for treating acute colitis [76]. Three pimarane-type diterpenoids-pedinophyllol K 133 (Figure 10), pedinophyllol L 134 (Figure 10), and libertellenone T 135 ( Figure 10)-were isolated from the endophytic fungal Phomopsis sp. S12 culture using the OSMAC strategy. The anti-inflammatory activities of all compounds were assessed using an LPS-induced inflammation model of mouse macrophage RAW264.7. Compound 135 dose-dependently inhibited the expression of inflammatory factors IL-1β and IL-6 at the mRNA level. Additionally, the anti-inflammatory activity of compounds 133-134 was similar to that of compound 135 in terms 0f IL-6 inhibition [77]. Two tetranorlabdane diterpenoids botryosphaerins G-H 136-137 ( Figure 10) were obtained from the ethyl acetate extract of Botryosphaeria sp. P483 isolated from the branches of the herb Huperzia serrata (Thunb.) Trev. and tested for their antifungal activities against Gaeumannomyces graminis, Fusarium solani, and Pyricularia oryzae by the disk diffusion method. Compound 137 showed effective antifungal activity at a concentration of 100 μg/disk with an inhibitory zone diameter of 9 mm. (The inhibitory zone diameter of positive control carbendazim was 15-18 mm.) Compounds 136-137 were evaluated for their nematicidal activities against Panagrellus redivivus and Caenorhabditis elegans and showed weak nematicidal activities, with 30% and 28% fatality rates at a 24h action concentration of 400 mg/L, respectively [78]. The isopimarane diterpene sphaeropsidin A 138 (Figure 10 [92]. Austin 179 ( Figure  12) was obtained from the ethyl acetate extract of Talaromyces purpurogenus H4 and Phanerochaete sp. H2 co-cultures, which showed moderate trypanocidal activity against T. cruzi at a concentration of 100 μg/mL, with an IC50 value of 36.6 μM. Notably, neither of the two endophytic fungi produced compound 179 when cultured separately under similar conditions [93].
To sum up, Meroterpenoids and their derivatives, which are mainly known for their antifungal properties in most plants species, have been found in endophytic fungi. However, recent studies have also reported anti-oxidative, anti-inflammatory, and anti-cancer activities from these compounds. Therefore, these microorganisms can be used in the development of drugs candidates for human, animal, and other agricultural activities. To sum up, Meroterpenoids and their derivatives, which are mainly known for their antifungal properties in most plants species, have been found in endophytic fungi. However, recent studies have also reported anti-oxidative, anti-inflammatory, and anti-cancer activities from these compounds. Therefore, these microorganisms can be used in the development of drugs candidates for human, animal, and other agricultural activities.

Lactones
Helicascolide F 180 ( Figure 13) was obtained from Talaromyces assiutensis JTY2 isolated from Ceriops tagal leaves. The cytotoxic activities of compound 180 against three human cancer cell lines (HeLa, MCF-7, and A549) were tested using an MTT assay, in which compound 180 showed a moderate cytotoxic effect on all tested cell lines, with an IC 50 value range of 14.1-38.6 µM [94]. Two β-lactones, polonicin A-B 181-182 (Figure 13), were obtained from the brown rice culture of the endophytic fungus Penicillium polonicum in the fruit of Camptotheca acuminata. Compound 181 showed effective glucose uptake activity at a concentration of 30 µg/mL on rat skeletal myoblast cell line L6, which enhanced 1.8-fold compared to that of the control. Compound 182 was used to assess its effect on GLUT4 translocation by using the fluorescent protein, IRAP-mOrange, which is stably expressed in L6 cells. It showed a 2.1-fold increase in fluorescence intensity on L6 cell membranes compared to the untreated controls [95]. The spirodilactone compound chaetocuprum 183 ( Figure 13) was obtained from cultures of the endophytic fungus Chaetomium cupreum of wild Anemopsis californica from New Mexico, U.S.A. Compound 183 showed a weak antimicrobial activity against S. aureus, with an MIC value of 50 µg/mL [96]. A phytotoxic bicyclic lactone, (3aS,6aR)-4,5-dimethyl-3,3a,6,6a-tetrahydro-2H-cyclopenta [b] furan-2one 184 (Figure 13), was obtained from the fermentation broth of Xylaria curta 92092022. Compound 184 contained a rare 5/5 rings-fusion system and was tested for antimicrobial activities against four pathogens (Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus NBRC 13276, Aspergillus clavatus F318a, and Candida albicans ATCC 2019) and the phytotoxicity against lettuce seedlings. Compound 184 showed moderate antimicrobial activities against Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus NBRC 13276 at a concentration of 100 µg/disk, with inhibitory zone diameters of 13 mm and 12 mm, respectively. At the concentration of 25 µg mL −1 , compound 184 showed 50% inhibition on lettuce roots with a root length of 1.6 ± 0.3 cm (3.2 ± 0.5 cm for the control). At a concentration of 200 µg mL −1 , compound 184 strongly inhibited lettuce seed germination, with 90% inhibition [97]. Lasiodiplactone A 185 (Figure 13) was obtained from the mangrove endophytic fungus Lasiodiplodia theobromae ZJ-HQ1 and contained a unique tetracyclic system (12/6/6/5) of RAL 12 (12-membered β-resorcylic acid lactone) with a pyran ring and a furan ring. Compound 185 showed significant anti-inflammatory activity by inhibiting the LPS-induced NO production in RAW 264.7 cells, with an IC 50 value of 23.5 µM, which was stronger than the positive control indomethacin (IC 50

Lactones
Helicascolide F 180 (Figure 13) was obtained from Talaromyces assiutensis JTY2 isolated from Ceriops tagal leaves. The cytotoxic activities of compound 180 against three human cancer cell lines (HeLa, MCF-7, and A549) were tested using an MTT assay, in which compound 180 showed a moderate cytotoxic effect on all tested cell lines, with an IC50   showed moderate insecticidal activities against the third-instar larvae of Helicoverpa armigera, with LC50 values of 0.72 mg/mL, 0.78 mg/mL, and 0.87 mg/mL, respectively. (The LC50 value for the positive control, matrine, was 0.29 mg/mL.) Additionally, the molecular mechanism of the insecticidal activity of compound 191 was investigated based on transcriptome sequencing. The identification of 5,732 differentially expressed genes was performed, of which 2,904 genes were downregulated and 2,828 genes were upregulated. The upregulated genes were primarily involved in cell autophagy, apoptosis, DNA mismatch repair, and replication [100]. A new quinone, identified as 1,3-dihydroxy-4-(1,3,4-trihydroxybutan-2-yl)-8-methoxy-9H-xanthen-9-one 192 (Figure 14), was obtained from Phomopsis sp. isolated from the rhizome of Paris polyphyllavar. in Yunnan, China. Compound 192 showed significant cytotoxic activities against A549 and PC3 cell lines, with IC50 values of 5.8 μM and 3.6 μM, respectively [101]. The anthraquinone derivative eurorubrin 193 ( Figure 14) was obtained from the ethyl acetate extract of the endophytic fungus Eurotium cristatum EN-220 of the seaweed Sargassum thunbergii and tested for its antimicrobial activities against three tested pathogens (E. coli, Physalospora obtuse, and Valsa mali), including its fatal activity against brine shrimp larvae. Compound 193 only showed a weak antimicrobial activity against E. coli, with an MIC value of 64 μg/mL. At the concentration of 10 μg/mL, compound 193 showed moderate fatal activity against brine shrimp larvae, with a fatality rate of 41.4% [102]. Isorhodoptilometrin-1-methyl ether 194 ( Figure  14  In summary, this review reported that fungal endophytes could produce Lactones and their derivatives through their metabolic activities. In addition, these compounds possessed biological activities, such as antimicrobial, anti-cancer, allelopathic, and antiinflammatory; thus, fungal endophytes that produce these compounds may be utilized in the pharmacological setup as alternatives to plant-derived compounds. (The LC 50 value for the positive control, matrine, was 0.29 mg/mL.) Additionally, the molecular mechanism of the insecticidal activity of compound 191 was investigated based on transcriptome sequencing. The identification of 5,732 differentially expressed genes was performed, of which 2,904 genes were downregulated and 2,828 genes were upregulated. The upregulated genes were primarily involved in cell autophagy, apoptosis, DNA mismatch repair, and replication [100]. A new quinone, identified as 1,3-dihydroxy-4-(1,3,4-trihydroxybutan-2-yl)-8-methoxy-9H-xanthen-9-one 192 (Figure 14), was obtained from Phomopsis sp. isolated from the rhizome of Paris polyphyllavar. in Yunnan, China. Compound 192 showed significant cytotoxic activities against A549 and PC3 cell lines, with IC 50 values of 5.8 µM and 3.6 µM, respectively [101]. The anthraquinone derivative eurorubrin 193 (Figure 14) was obtained from the ethyl acetate extract of the endophytic fungus Eurotium cristatum EN-220 of the seaweed Sargassum thunbergii and tested for its antimicrobial activities against three tested pathogens (E. coli, Physalospora obtuse, and Valsa mali), including its fatal activity against brine shrimp larvae. Compound 193 only showed a weak antimicrobial activity against E. coli, with an MIC value of 64 µg/mL. At the concentration of 10 µg/mL, compound 193 showed moderate fatal activity against brine shrimp larvae, with a fatality rate of 41.4% [102]. Isorhodoptilometrin-1-methyl ether 194 (Figure 14), emodin 195 (Figure 14), and 1-methyl emodin 196 ( Figure 14) were obtained from cultures of the endophytic fungus Aspergillus versicolor of the red seaweed Halimeda opuntia. Compounds 194-196 were evaluated for their inhibiting activities against the hepatitis C virus NS3/4A protease, where Compounds 195-196 showed weak inhibition activities, with IC 50 values ranging from 22.5 to 40.2 µg/mL [103]. The quinone altersolanol A 197 (Figure 14) was isolated from the endophytic fungus Stemphylium globuliferum of the medicinal plant Mentha pulegium (Lamiaceae). Compound 197 inhibited the proliferation of K562 and A549 cells in a time-dependent, dose-dependent manner and caused apoptosis by cleaving Caspase-3 and Caspase-9 and decreasing anti-apoptotic protein expression [104]. with IC50 values ranging from 22.5 to 40.2 μg/mL [103]. The quinone altersolanol A 197 ( Figure 14) was isolated from the endophytic fungus Stemphylium globuliferum of the medicinal plant Mentha pulegium (Lamiaceae). Compound 197 inhibited the proliferation of K562 and A549 cells in a time-dependent, dose-dependent manner and caused apoptosis by cleaving Caspase-3 and Caspase-9 and decreasing anti-apoptotic protein expression [104]. Anthraquinones, quinones, and related glycosides are known for their anti-viral and anti-apoptotic activity both in vitro and in vivo. Interestingly, these compounds have been identified and isolated from fungal endophytes by various studies and have similarly shown anti-viral and anti-apoptotic activities. Thus, endophytes that produce these compounds may serve as cheap and environmentally friendly alternative sources for the development of antimicrobial drugs instead to plant sources.   Anthraquinones, quinones, and related glycosides are known for their anti-viral and anti-apoptotic activity both in vitro and in vivo. Interestingly, these compounds have been identified and isolated from fungal endophytes by various studies and have similarly shown anti-viral and anti-apoptotic activities. Thus, endophytes that produce these compounds may serve as cheap and environmentally friendly alternative sources for the development of antimicrobial drugs instead to plant sources. To summarize, endophytic fungi are alternative sources of steroids and their derivatives; thus, they may be harnessed for the production of various drugs since they have shown antimicrobial and anticancer activity in previous studies.   To summarize, endophytic fungi are alternative sources of steroids and their derivatives; thus, they may be harnessed for the production of various drugs since they have shown antimicrobial and anticancer activity in previous studies.   [108]. The indene derivative methyl 2-(4-hydroxybenzyl)-1,7-dihydroxy-6-(3-methylbut-2-enyl)-1H-indene-carboxylate 207 (Figure 16) obtained from the endophytic fungus Aspergillus flavipes Y-62 isolated from Suaeda glauca Bunge in Zhoushan, Zhejiang, China, showed weak antimicrobial activities against Pseudomonas aeruginosa, Klebsiella pneumonia, and Staphylococcus aureus, with MIC values ranging from 32 to 128 µg/mL [109]. The polychlorinated triphenyl diether simatorone 208 (Figure 16) was isolated from Microsphaeropsis sp. cultures, and its antimicrobial activities against three pathogens (Escherichia coli, Bacillus megaterium, and Microbotryum violaceum) were evaluated using an agar diffusion assay. Compound 208 showed effective antimicrobial activities against B. megaterium and E. coli with inhibitory zone diameters of 14 mm and 18 mm, respectively [110]. Two alkylated furan derivatives-5-(undeca-3 ,5 ,7trien-1 -yl) furan-2-ol 209 ( Figure 16) and 5-(undeca-3 ,5 ,7 -trien-1 -yl) furan-2-carbonate 210 ( Figure 16)-were obtained from the methanol extract of the endophytic fungus Emericella sp. XL029 isolated from Panax notoginseng leaves in Hebei, China. Compounds 209-210 both showed potent antifungal activities against six tested plant pathogenic fungi (Rhizoctorzia solani, Verticillium dahliae Kleb, Helminthosporium maydis, Fusarium oxysporum, Fusarium tricinctum, and Botryosphaeria dothidea), with MIC values ranging from 25 to 3.1 µg/mL [111]. The new azaphilone, isochromophilone G 211 (Figure 16), was obtained from the endophytic fungus Diaporthe perseae sp. isolated from Pongamia pinnata (L.) Pierre. Compound 211 showed significant DPPH and ABTS radical scavenging activities, with IC 50 values of 7.3 µmol/mL and 1.6 µmol/mL, respectively [112]. The furan derivative, 3-(5-oxo-2,5-dihydrofuran-3-yl) propanoic acid 212 (Figure 16), was obtained from the endophytic fungus Aspergillus tubingensis DS37 isolated from Decaisnea insignis (Griff.) Hook & Thomson, and showed significant inhibition activities against Fusarium graminearum and Streptococcus lactis, with MIC values of 16 µg/mL and 32 µg/mL, respectively [113]. The pyrrolidinone derivative, nigrosporamide A 213 (Figure 16), was isolated from the endophytic fungus Nigrospora sphaerica ZMT05 of Oxya chinensis Thunberg and showed a three-fold higher α-glucosidase inhibition activity than the positive control acarbose (IC 50 value of 446.7 µM) with an IC 50 value of 120.3 µM. Compound 213 has the potential to be a lead compound for the development of α-glucosidase inhibitors [114]. The production of the terrein derivative asperterrein 214 ( Figure 16) was induced by co-culturing endophytic fungi Aspergillus terreus EN-539 and Paecilomyces lilacinus EN-531 of the marine red alga Laurencia okamurai. Compound 214 showed weak antimicrobial activities against Physalospora piricola and Staphylococcus aureus, with MIC values ranging from 32 to 64 µg/mL. Additionally, compound 214 was not detected in the sterile cultures of the two fungi alone [115]. The endophytic fungus Lachnum palmae of Przewalskia tangutica was isolated to halogenated dihydroisocoumarins palmaerones A-F 215-220 ( Figure 16) under the guidance of UPLC-ESIMS. The antimicrobial activities of all compounds against five tested pathogens (Cryptococcus neoformans, Penicillium sp., Candida albicans, Bacillus subtilis, and Staphylococcus aureus) were evaluated using the broth microdilution method. Compounds 215-220 showed potent to weak antimicrobial activities against all tested pathogens, with MIC values ranging from 10 to 55 µg/mL. Additionally, compounds 215 and 219 showed moderate inhibition of LPS-induced NO production in RAW264. All the information about the new compounds have been summarized below in Table  2. Over the past few years, plants have been a major source of numerous compounds that possess biological activities; however, this review revealed that most of these compounds were also produced by various endophytes, especially fungi. Therefore, the isolation and development of these compounds as novel drug candidates would be of great importance to the pharmacological industry since endophytes are easy to manage, keep, and work with compared with plants. Thus, we conclude that endophytic fungi may serve as alternative sources of bioactive compounds of pharmacological interest.

Other Types of Compounds
All the information about the new compounds have been summarized below in Table 2.

Future Prospects and Challenges of Using Endophytic Fungi as an Alternative Source of Plant Bioactive Compounds
Endophytic fungi are hidden and subtle dwellers in several plant tissues and intercellular spaces and can produce diverse chemical structures and efficient, low-toxic new secondary metabolites that were initially thought to be produced by the host plants. The current reports on the biosynthesis of plant metabolites by endophytic fungi, in conjunction with recent research advances in fermentation culture, extraction, isolation, and structure identification techniques, permit us to rapidly uncover new valuable compounds. Generally, fungi are chemically diverse, easily cultured, and biologically active modalities that have great flexibility to be regulated by adding precursors, elicitors, and specific enzymes to effectively increase the quantity and yield of bioactive compounds. Table 3 represents the culture conditions and specific bioactive secondary metabolites and yields produced by various endophytic fungi. Endophytic fungi can convert active compounds of the host plant into more potent derivatives. This makes endophytic fungi an alternative and sustainable source of plant bioactive compounds [117,118]. The search for new compounds in endophytic fungi requires specific theories and ingenious bioprospecting strategies. Along with the continuously growing literature reports, the most promising host plants can be selected. It includes the selection of (A) plants from special habitats or growing in biodiversity-rich areas, including mangrove plants in tropical marine intertidal zones, and (B) medicinal and indigenous plants with ethnopharmacological uses, including Camptotheca acuminata and Ageratina adenophora. These selection criteria provide a reference for the current and future screening of host plants for endophytic fungi with new bioactive compounds [119,120]. This review has summarized 220 new compounds obtained between 2011 and 2021 from endophytic fungi using different culture methods, including the common culture, co-culture with bacteria or other fungi, and the addition of metal ions. These new compounds have unique molecular structures, and these rare structures allow these compounds to possess diverse biological activities, including significant antimicrobial and cytotoxic activities and α-glucosidase inhibition. These compounds have the potential to be modified as pro-drug molecules or directly developed as drugs for treating certain diseases. However, most of the current studies on the activity of new compounds with endophytic fungal sources are limited to in vitro studies; therefore, animal experiments and human intervention clinical trials are needed to further investigate the in vivo activities and mechanisms of action of the new compounds.
Unfortunately, endophytic fungi as new sources of bioactive secondary metabolites encounter various limitations, including the attenuated yield of secondary metabolites due to long-term storage and repeated passages under laboratory culture conditions, silencing of biosynthetic gene clusters or low level of expression (activation of gene clusters depends on environmental factors). Thus, the ability of endophytic fungi to produce new compounds of interest has been underestimated [129]. The expression could be upregulated by physicochemical and genetic manipulation techniques to increase the production of specific metabolites in endophytic fungi and to produce analogs of new active secondary metabolites. Methods including the OSMAC strategy (activation of silent biosynthetic gene clusters mediated by changes in medium composition, temperature, and aeration efficiency to produce desired metabolites), co-culture (mimicking natural ecosystems and triggering silent gene clusters to promote metabolite secretion and enhance bioactive metabolite production by microbial interaction-induced stress responses), and chemical epigenetic modification methods have been used to isolate new compounds. It was found that the addition of micromolar or even nanomolar small-molecule chemicals to cultures inhibits or activates relevant enzymes and remodels the fungal epigenome to increase the diversity of its secondary metabolites, including DNA methyltransferases (DNMTs) and histone deacetylase inhibitors (HDACs) [130,131]. The addition of epigenetic modifiers (5 µM SAHA and 10 µM AZA) to the endophytic fungus Xylaria psidii isolated from leaves of Vitis vinifera showed elevated resveratrol concentrations of 52.32 µg/mL and 48.94 µg/mL, respectively, by HPLC analysis (control concentration was 35.43 µg/mL). The treatments with 5 µM SAHA and 10 µM AZA showed stronger antioxidant activity with 30.92% and 33.82% DPPH radical scavenging, respectively, compared to the wild strain (19.26%) [132]. Unlike the chemical epigenetic modification methods reported, introducing exogenous substances as precursors into the cultures, including methyl jasmonate, causes the production of new compounds containing their structural units [133]. However, the addition of host plant components to the culture to induce the production of new compounds has rarely been reported. Additionally, it is necessary to elucidate the pathways by which endophytic fungi biosynthesize secondary metabolites, including the enzymes and genes involved via "omics" techniques-genomics, transcriptomics, and metabolomics-in regulating and manipulating the biosynthetic process to increase the number of new compounds [134].

Conclusions
Pharmaceutical chemists are turning their focus on the development of safe, efficient, and low-toxic new drugs from natural sources. Endophytic fungi may serve as renewable sources of novel bioactive compounds with pharmacological activities, as the number of new compounds to be isolated in the future tends to increase exponentially and rapidly. In addition, numerous studies have also reported that these bioactive compounds isolated from the endophytic fungi are also present in plants and have similar biological activities as the compounds from plant sources. Therefore, we conclude that endophytic fungi may be the best alternative for harnessing pharmacological bioactive compounds for the development of drugs for both human and animal use. Hence, there is a need for the identification of more compounds with pharmacological activity from endophytic